Abstract: Clinically, resistant breast cancer cells can enter a dormant phase following initial breast cancer treatment, with patients developing recurrence many years later. There is also increasing evidence that breast tumor recurrences arise from only a small subset of BCSC, whose in vivo behavior overlaps with the dormant sub- population. Dormant BCSCs may be highly resistant to traditional anti-cancer therapy. Thus, there is a pressing need to identify tumor specific-antigens expressed by dormant cancer stem cells and to confirm that immunotherapy targeting these antigens can mediate tumor regression and eliminate dormant cancer cells. Our goal is to develop a novel strategy for the treatment of breast cancer based on a new monoclonal antibody (mAb), which preferentially reacts with the bulk, non-proliferating breast cancer cells and breast cancer stem cells (BCSC). We have previously observed that adoptive transfer of T cells that were primed by BCSCs could efficiently inhibit spontaneous tumor progression. Furthermore, post-treatment serum immunoglobulin was found to bind neuneg-variant C2D1 breast tumor cells. To characterize antigenic targets, we obtained a hybridoma specific for this monoclonal antibody (mAb4; IgM) by fusing spleen cells from BCSC-immunized mice with myeloma cells. mAb4 specifically bound to non-proliferating adherent C2D1 and C2D1-BCSC cells, but not normal mammary gland cells or NIH 3T3 cells. Moreover, mAb4 induced strong complement-mediated cytotoxicity against target tumor cells. These findings suggest that the antigen recognized by mAb4 may be a useful target against dormant breast cancer cells. In this proposal, we will focus on the identification of the target antigen recognized by mAb4 antibody using SEREX method. In addition, our data showed that mAb4 also cross-reacted with human BCSC cells. Therefore, we anticipate that mAb4 may also target homologous antigen(s) in human breast tumor. The identification of a tumor-associated antigen for dormant cancer cells may impact the development of novel therapeutics. In addition, we will also test the hypothesis that mAb4 may be effective in preventing primary breast tumor development in neuN transgenic mice, and that mAb4 combined with chemotherapy may be therapeutically efficacious for eradicating established tumors and preventing recurrence. To accomplish the overall objective, two specific aims will be pursued. Specific aim 1 will identify the antigen recognized by mAb4 antibody and to determine the homologous antigen in human breast cancer. Specific aim 2 will investigate whether mAb4 alone or combined with chemotherapy can eliminate spontaneous breast tumor in neuN transgenic mice in situ.